KR20210135234A - Cell carrier particulate agglomerate and method for preparing same - Google Patents

Abstract

The present disclosure is a cell carrier particulate aggregate and a method for preparing the same. This cell carrier particulate aggregate is formed of an aggregate of cell carrier particles and has a specific shape, including flakes and blocks. The manufacturing method of the cell carrier particle aggregate is stamp forming, mold forming, freeze drying or dehydration and evaporation,

Description

Cell carrier particulate aggregate and method for preparing same

The present invention relates to an aggregate of cell carrier particles and a method for preparing the same.

Cell carrier particles (typically 1-1000 um in diameter) refer to microbeads that can be used for growth or co-culture of adherent cells. Currently, all commercially available microcarriers are used, stored and packaged in powder form. However, since microcarriers are micron-scale microbeads, they tend to generate a large amount of static electricity in a dry powder state. Therefore, it can easily cause leakage of the microcarrier powder during use or transportation, decrease in effective mass, as well as contamination of the surrounding environment and the product itself. In addition, it is difficult to store these powdered microcarriers, since their nature leads to such limitations, which still result in a large amount of unnecessary loss, no matter what kind of packaging is used. Furthermore, many measuring instruments such as auxiliary instruments are needed in various processes, such as weighing, which are very time-consuming and wasteful of effort, so it is difficult to use them. Therefore, in order to facilitate use in cell culture, it is necessary to provide an aggregate of cell carrier particles.

It is an object of the present invention to provide an aggregate of cell carrier particles and a method for preparing the same. In the present invention, the cell carrier particles in the powder state are aggregated and shaped so that the aggregate is formed. These aggregates are microcarriers during use, packaging and transport, such as generating large amounts of static electricity, easy to shed during use and weighing, electrostatic absorption on the walls of containers or implements, difficulties in operation, causing losses, and the like. Avoid problems caused by powder. In addition, it is advantageous to meet the requirements in the quantitative use and sterilization of microcarriers in the cell culture process.

As used herein, “cell carrier particle” refers to microcarriers having a diameter of 1 to 1000 μm prepared from synthetic polymers and/or natural biopolymers. Synthetic polymers include at least polyethylene glycol, polyethylene glycol derivatives, polyethylene glycol diacrylate (PEGDA4000), polypropylene (ollypropylene), polystyrene , polyacrylamide, polylactic acid, poly(hydroxy acid) ((poly(hydroxy acid)), poly(lactic-co-glycolic acid) ((poly(lactic-co-) glycolic acid)), polydimethylsiloxane, polyanhydride, poly(acid ester) ((poly(acid ester)), polyamide, poly(amino acid) ((poly(amino acid) acid)), polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate (polycarbonate), and one selected from the group consisting of polyethylene oxide (polyethylene oxide) Natural biopolymer is at least collagen (collagen), proteoglycan (proteoglycan), glycoprotein (glycoprotein), gelatin (gelatin), gelatin derivatives (gelatin derivatives), alginate, alginate derivatives, agar, matrigel, hyaluronic acid, laminin, One selected from the group consisting of fibronectin, or tissue decellularized materials.

The cell carrier particle aggregate provided in the present invention is formed by aggregating cell carrier particles and has a specific shape. These specific shapes include tablet shapes and block shapes. The tablet or block may have a cross-section of a circle, cylinder, square, diamond-shaped, triangular, oval, concave or convex polyhedron, or the like. The aggregate of cell carrier particles has very good rehydration properties and excellent dispersibility properties upon rehydration.

The cell carrier particle aggregate according to the present invention can be produced by the following method: Cell carrier particles are aggregated and shaped under an external force to obtain a cell carrier particle aggregate.

In particular, the cell carrier particles can be assembled and shaped in a punch-forming process.

The punch-forming process is carried out under the following conditions:

Punch molds are formed from a beveled flat punching mold, a shallow arc punching mold, a deep arc punching mold, or a full flat punching mold. is selected;

The punch-forming machine is characterized in that the upper punch has an adjustable range of 0 to 50 mm, and the lower punch has an adjustable range of 0 to 50 mm;

The pressure range is from 0 to 200 KN, which may be selected according to actual needs in order to prepare an aggregate of cell carrier particles having an appropriate hardness; and

In particular, an automatic punch-forming machine can be used, in which each tablet of the cell carrier particle aggregate can be obtained automatically after the motor rocker rotates once.

For punch-forming machines, the mass and weight of a material is determined by measuring its volume.

In particular, the cell carrier particles can be assembled and shaped by a mold-forming process.

The mold-forming process comprises the following steps:

mixing the cell carrier particles with water or a volatile organic solvent and filling the resulting mixture into a mold; and then the mold is placed in an oven and dried to obtain an aggregate of cell carrier particles;

wherein water or an organic solvent may be added in an amount of 5 to 100 times the mass of the cell carrier particles;

The organic solvent may be a solvent such as methanol, ethanol, tert-butanol, or isopropanol; and

The temperature of the oven can be 30-200° C., and drying can be carried out for a period of 12-96 hours, and, thereby, a solid and stable aggregate of cell carrier particles with a uniform unit mass and the same shape. can get

In particular, the cell carrier particles can be collected and shaped by a lyophilization process.

The lyophilization process comprises the following steps:

mixing the cell carrier particles with water or a volatile organic solvent and shaping the resulting mixture into a specific shape, then freezing the molded mixture to obtain a frozen mixture; and freeze-drying the frozen mixture to obtain an aggregate of cell carrier particles;

wherein water or an organic solvent may be added in an amount of 5 to 100 times the mass of the cell carrier particles;

The organic solvent may be a solvent such as methanol, ethanol, tert-butanol, or isopropanol solvent;

The molded mixture is frozen in a freezing container at a low temperature ranging from -196°C to 0°C to obtain a frozen mixture; and

The frozen mixture is lyophilized in a lyophilizer for 2 to 96 hours to obtain an aggregate of cell carrier particles.

In particular, the cell carrier particles can be aggregated and shaped in a dehydrating-evaporating process;

wherein the dehydration-evaporation process comprises the following steps;

mixing the cell carrier particles with water or a volatile organic solvent and shaping the mixture into a specific shape; and then dehydrating and evaporating the shaped mixture to obtain an aggregate of cell carrier particles;

wherein water or an organic solvent may be added in an amount of 5 to 100 times the mass of the cell carrier particles;

The organic solvent may be a solvent such as methanol, ethanol, tert-butanol, or isopropanol solvent;

Through a natural dehydration process at room temperature, such as volatilization for 1 to 7 days at room temperature, a robust and stable cell carrier particle aggregate with a regular shape can be obtained; and

For example, by drying in an oven, such as drying in an oven for 2 to 96 hours, a firm and stable cell carrier particle aggregate having a regular shape can be obtained.

1 is a photograph of cell carrier particles in Example 1 of the present invention.
2 is a photograph of punch-formed cell carrier particles in Example 1 of the present invention.
3 shows the initial state of the cell carrier particle aggregate when dispersed in a liquid in Example 1 of the present invention.
4 shows the final state of the cell carrier particle aggregate after being completely dispersed in the liquid in Example 1 of the present invention.
5 is an SEM photograph of cell carrier particles in Example 1 of the present invention.
6 is an SEM photograph of the cell carrier particle aggregate in Example 1 of the present invention.
7 is an SEM photograph of particles obtained after dispersing the cell carrier particle aggregate in a liquid in Example 1 of the present invention.
8 is a photograph showing cell carrier particles filled in a cylindrical assembly frame in Example 2 of the present invention.
9 is a photograph showing mold-formed cell carrier particles in Example 2 of the present invention.
10 is a photograph of the cell carrier particle aggregate after being dispersed in a liquid in Example 2 of the present invention.
11 is a photograph of the cell carrier particle aggregate after absorbing water in Example 3 of the present invention.
12 is a photograph of lyophilization-formed cell carrier particles in Example 3 of the present invention.
13 is a photograph of the cell carrier particle aggregate after being dispersed in a liquid in Example 3 of the present invention.
14 is a photograph of the cell carrier particle aggregate after absorbing water in Example 4 of the present invention.
15 is a photograph of cell carrier particles formed after the dehydration-evaporation process in Example 4 of the present invention.
16 is a photograph of the cell carrier particle aggregate after being dispersed in a liquid in Example 4 of the present invention.

Example

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, materials, reagents, and the like used in the following examples are all commercially available.

Each individual cell carrier particle used in the following examples was prepared as follows.

Preparation of reaction solution: PEGDA4000 powder was dissolved in DPBS solution at room temperature to obtain a pre-polymerized solution; The pre-polymerization solution is then thoroughly mixed with ammonium persulfate and N,N,N',N'-tetramethyl ethylenediamine (N,N,N',N'-tetramethyl ethylenediamine) to form the reaction solution. obtained, wherein the concentration of PEGDA4000 in the reaction solution was 10 g/100 ml, the concentration of ammonium persulfate in the reaction solution was 0.5 g/100 ml, and N,N,N′,N′-tetramethyl ethylenediamine in the reaction solution The concentration was 0.05 g/100 ml.

Preparation of each cell carrier particle with an arrayed mold: A PMMA mold with holes in an array format, already prepared, is placed in an ice bath, and 200 ul of the prepared PMMA mold is placed in an ice bath. The reaction solution was uniformly added dropwise to the PMMA mold, and then, using a cover glass, the reaction solution on the surface of the mold was scraped slowly and uniformly into the holes of the mold. The mold filled with the PEGDA reaction solution was frozen at -20 °C for 20 hours. After completion of the reaction, the mold was transferred to a freeze dryer (-50° C., 20 pa) and dried for 30 minutes to obtain white porous cell carrier particles with a diameter of 1-1000 um.

Example 1. Punch-forming process

A punch-forming machine is an automatic punch-forming machine in which a tablet product is obtained after one turn of a motor rocker. For punch-forming machines, the mass and weight of the material are determined by measuring its volume.

20 mg of cell carrier particles were first weighed on a balance, this weighed material was then poured into the feed port of a punch-forming machine, and an inclined flat punch mold was selected. The upper punch of the punch-forming machine was adjusted in the range of 0 to 50 mm, and the lower punch of the punch-forming machine was adjusted in the range of 0 to 50 mm. The handle of the punch-forming machine was operated to fill the mold with material and the pressure was adjusted in the range of 0 to 200 KN. After turning on the motor and turning the motor rocker once, one punch-forming operation is finished. The automatic punch-forming machine can continuously perform punch-forming operations at a constant speed, wherein the punch-forming speed is 1 to 10 seconds per tablet. After the punch-forming process was completed, the assembly was placed in a pre-prepared container.

A photograph of the aggregate formed in this example is shown in FIG. 2 , and FIG. 1 is a photograph of the powdered cell carrier particles before this morphogenesis process.

In the method of this embodiment, the unit mass of the assembly can be adjusted by adjusting the height of the lower punch, ie by adjusting the volume of the material.

During the manufacturing process, it is necessary to pay attention to the following operations:

(1) When performing the punch-forming operation, the punch-forming strength must be increased in order to compress the tablet, so that the assembled tablet is not broken by a small impact (by adjusting the height of the upper punch, the punch-forming strength is can be increased; here turn left to decrease height and right turn to increase height);

(2) When adjusting the mass of the tablet-like assembly, the rocker must be held vertically ^{90° upwards in the horizontal plane;}

(3) when performing the punch-forming operation, it should be observed whether the mold is filled with material or not; If not, turn the handle in the opposite direction and fill the mold with material to ensure that the mass of each tablet is correct; and

(4) In the punch-forming machine, since the mass of a material is determined by measuring its volume, it is necessary to change the mold in order to obtain a product having a desired mass by the punch-forming process. The mass can be adjusted within the range of 1 mg to 1000 mg.

The assembly prepared in this example was placed in a container and the container was then placed on a shaker that was continuously shaken. The rotational speed of the shaker ranged from 0 to 1000 rpm, and the agglomerates were not brittle or similar after continuous shaking for 96 hours. It can be seen that the aggregate manufactured in this example is stable in shape and not easily broken, and that the unit mass is accurate and can be adjusted.

After adding a very small amount of liquid to the aggregates prepared in this example, the aggregates dispersed quickly to form separate carrier supports without any change in physical and chemical properties. 3 shows the initial state of the aggregate when dispersed in a liquid, and FIG. 4 shows the final state of the aggregate after being completely dispersed in the liquid. 5 is an SEM photograph of cell carrier particles before punch-forming process; 6 is an SEM photograph of the cell carrier particle aggregate after the punch-forming process; 7 is an SEM photograph of powdered cell carrier particles after punch-forming process, dispersion in liquid, and lyophilization. By comparing the SEM photographs representative of these three states, after the punch-forming process, the cell carrier particles still retain their properties, such as spherical shape, pore connectivity, and dispersing ability, compared to before the punch-forming process. It can be seen that this can be advantageous for the subsequent adherent cell culture.

The cell carrier particle aggregate prepared in this example is convenient for packaging and transport, and it is possible to avoid static electricity resulting from powder production and loss therefrom. The assembly may be individually packaged and sterilized, which is convenient for aseptic operation in cell culture. Additionally, the aggregate can be quantitatively shaped and is therefore convenient to use.

Example 2. Mold-forming process

To a fixed amount of powdered cell carrier particles, 50 times the weight of cell microcarriers was added with deionized water. Using the aid, the resulting mixture was equally filled into the mold. The frame was cylindrical (it could also be round, square, diamond-shaped, triangular, oval, or concave or convex polyhedron, etc.). A mold (as shown in Fig. 8) containing a certain amount of cell carrier particles was placed in an oven at 60 °C and dried continuously for 96 hours, as shown in Fig. 9, a firm and stable with uniform unit mass and the same shape. Cell carrier particles were obtained.

When the cell carrier particle aggregate prepared in this Example was added to water, the aggregate could be rapidly dispersed, as shown in FIG. 10, to form separated cell carrier particles. These results show that the appearance and physical properties of the particles remained unchanged.

The cell carrier particle aggregate prepared in this Example can rapidly absorb water and can be dispersed after contact with a liquid, which will be advantageous for the subsequent adherent cell culture.

After comparing the SEM pictures of the cell carrier particles before and after the forming process as well as after rehydration and dispersion in liquid, in this example the cell carrier particles after the mold-forming process were punch-forming It can be seen that compared with those before processing, their properties such as spherical appearance, porous connectivity, and dispersion were maintained.

The cell carrier particle aggregate prepared in this example is convenient for packaging and transport, and it is possible to avoid static electricity resulting from powder production and loss therefrom. The assembly may be individually packaged and sterilized, which is convenient for aseptic operation in cell culture. Additionally, the aggregate can be quantitatively shaped and is therefore convenient to use.

Example 3. Freeze-dried forming process ( Lyophilization forming process)

To a fixed amount of powdered microcarriers, 100 times the weight of cell microcarriers was added with deionized water. Using the aid, the resulting mixture was assembled into blocks having a regular shape, as shown in FIG. 11 . This aggregate was frozen in a freezing vessel at a low temperature ranging from -196 °C to 0 °C. The frozen aggregates were lyophilized in a lyophilizer for 96 hours; After taking out, lyophilized aggregates were formed as shown in FIG. 12 .

The cell carrier particle aggregate prepared in this Example can rapidly absorb water and can be dispersed after contact with a liquid, which will be advantageous for the subsequent adherent cell culture.

After comparing the SEM pictures of the cell carrier particles before and after the forming process, as well as after rehydration and dispersion in a liquid, in this example cell carrier particles after the lyophilization forming process , maintained their properties such as spherical appearance, porous connectivity, and dispersion when compared with those before the punch-forming process.

The cell carrier particle aggregate prepared in this example is convenient for packaging and transport, and it is possible to avoid static electricity resulting from powder production and loss therefrom. The assembly may be individually packaged and sterilized, which is convenient for aseptic operation in cell culture. Additionally, the aggregate can be quantitatively shaped and is therefore convenient to use.

Example 4. Dehydrating-evaporating forming process

To a fixed amount of powdered microcarriers, 20 times the weight of cell microcarriers was added with deionized water. Using an auxiliary tool, the resulting mixture was formed into blocks having a regular shape, as shown in FIG. 14 . These cellular microcarrier blocks with constant mass were naturally dehydrated and evaporated at room temperature. After volatilization for 7 days, robust and stable cell microcarrier aggregates with regular shapes were obtained, as shown in FIG. 15 .

When the cell carrier particle aggregate prepared in this Example was put into water, the aggregate was quickly dispersed to form separated microcarrier particles, as shown in FIG. 16 . These results show that the appearance and physical properties of these particles remained unchanged.

The cell carrier particle aggregate prepared in this Example can rapidly absorb water and can be dispersed after contact with a liquid, which will be advantageous for the subsequent adherent cell culture.

After comparing the SEM pictures of the cell carrier particles before and after the forming process, as well as after rehydration and dispersion, the cell carrier after the dehydrating-evaporating forming process in this example It can be seen that the particles maintained their properties such as spherical appearance, porous connectivity, and dispersion when compared to those before the punch-forming process.

The cell carrier particle aggregate prepared in this example is convenient for packaging and transport, and it is possible to avoid static electricity resulting from powder production and loss therefrom. The assembly may be individually packaged and sterilized, which is convenient for aseptic operation in cell culture. Additionally, the aggregate can be quantitatively shaped and is therefore convenient to use.

industrial application

The present invention achieves the following advantageous effects.

In the cell carrier particle aggregate according to the present invention, the cell carrier particle has porous connectivity, has greater elasticity, and is resistant to extrusion. After pressing carrier particles into aggregates, these carrier particles can be rapidly dispersed into separate carrier supports with the addition of very small amounts of liquid, without changing their physical and chemical properties. After comparing SEM photographs of the cell carrier in powder form before the aggregation and molding step, the cell carrier after assembling into a tablet-like aggregate, and the cell carrier after the lyophilized aggregate absorbs and disperses water, the punch-forming process It could be seen that the cell carrier particles after that maintained their properties such as a spherical shape, porous connectivity, and dispersibility compared to those before the punch-forming process, which is advantageous for subsequent adherent cell culture. Therefore, the aggregate of the cell carrier particles according to the present invention has very strong rehydration properties and good dispersibility upon rehydration, and the physical properties of the cell carrier particles remain unchanged before and after the aggregation.

Since there is no powder on the surface of the aggregate obtained by forming processes without the addition of other components, static electricity is reduced as much as possible. The product is aesthetic and convenient to use, thereby avoiding the static electricity and consequent losses resulting from the powdered product.

Cell carrier particle aggregates according to the present invention may be quantified, sterilized and stored in single tablet form in pharmaceutical blister packaging, or may be quantified, sterilized and stored in multiple tablet form in vials. Such individual packaging and sterilization would be convenient for aseptic manipulation in cell culture.

Claims (13)

An aggregate of cell carrier particles, wherein the aggregate of cell carrier particles is formed by aggregating cell carrier particles.
According to claim 1,
An aggregate of cell carrier particles, characterized in that the aggregate of the cell carrier particles has a specific shape.
3. The method of claim 2,
the particular shape includes a tablet shape and a block shape; and
The aggregate of cell carrier particles, characterized in that it has excellent rehydration property and good dispersibility upon rehydration.
A method for preparing an aggregate of cell carrier particles according to any one of claims 1 to 3, comprising:
Aggregating and shaping the cell carrier particles under an external force to obtain an aggregate of the cell carrier particles.
5. The method of claim 4,
wherein the cell carrier particles are aggregated and shaped by a punch-forming process.
6. The method of claim 5,
A method, characterized in that the punch-forming process is carried out under the following conditions:
A punching mold may be a beveled flat punching mold, a shallow arc punching mold, a deep arc punching mold, or a full flat punching mold. flat punching molds);
The punch-forming machine has an upper punch adjusted in a range between 0 and 50 mm and a lower punch adjusted in a range between 0 and 50 mm; and
A method, characterized in that the pressure range is between 0 and 200 KN.
5. The method of claim 4,
The method of claim 1, wherein the cell carrier particles are aggregated and shaped by a mold-forming process.
8. The method of claim 7,
A method, characterized in that the mold-forming process comprises the steps of:
mixing the cell carrier particles with water or a volatile organic solvent and filling the resulting mixture into a mold; and then placing the mold in an oven and drying to obtain an aggregate of cell carrier particles.
9. The method of claim 8,
said water or organic solvent is added in an amount of 5 to 100 times the mass of the cell carrier particles: and
The temperature of the oven is 30 to 200 °C, and the drying is carried out for a period of 12 to 96 hours.
5. The method of claim 4,
A method, characterized in that the cell carrier particles are aggregated and shaped by a lyophilization process;
Here, the lyophilization process includes the following steps: mixing the cell carrier particles with water or a volatile organic solvent and shaping the resulting mixture into a specific shape, followed by shaping to obtain a frozen mixture freezing the mixture; and freeze-drying the frozen mixture to obtain an aggregate of the cell carrier particles.
11. The method of claim 10,
The water or organic solvent is added in an amount of 5 to 100 times the mass of the cell carrier particles:
the molded mixture is frozen into a freezing container at a low temperature ranging from -196° C. to 0° C. to obtain the frozen mixture; and
The method of claim 1, wherein the frozen mixture is freeze-dried in a freeze dryer for a period of 2 to 96 hours to obtain an aggregate of the cell carrier particles.
5. The method of claim 4,
a method wherein the cell carrier particles are aggregated and shaped by a dehydrating-evaporating process;
wherein the dehydration-evaporation process comprises the following steps:
mixing the cell carrier particles with water or a volatile organic solvent and shaping the mixture into a specific shape; and thereafter dehydrating and evaporating the shaped mixture to obtain an aggregate of the cell carrier particles.
13. The method of claim 12,
said water or organic solvent is added in an amount of 5 to 100 times the mass of said cell carrier particles: and
The method according to claim 1, wherein the cell carrier particles are aggregated and shaped by a method of natural dehydration at room temperature or a method of drying in an oven.

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